Human Biliverdin Reductase Is Autophosphorylated, and Phosphorylation Is Required for Bilirubin Formation

Abstract

Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity. An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein. Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated. Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent. In addition, 2(3)-O-(2,4,6-trinitrophenyl)adenosine 5-triphosphate fluorescence titration of BVR gave a lower K d at pH 8.7 than at pH 7.4 (15.5 versus 28.0 M). Mn 2؉ was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60°C for 10 min. The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase. Potato acid phosphatase treatment reversibly inactivated the enzyme. The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition. Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate. The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H 2 O 2 . The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.

Crystal Structure of CC3 (TIP30)

“…An alternative way of rationalizing putative kinase and reductase activities for CC3 is a dual function involving the ability to bind ATP and NADPH for the different reactions. Whereas such a dual specificity appears unlikely, it may be noted that the SDR biliverdin IX␣ reductase (28), in addition to catalyzing a reductase reaction, has been reported to have protein kinase activity and be capable of autophosphorylation. Based on our current report, we find no evidence for ATP binding to CC3 in thermal shift and co-crystallization studies.…”

“…The ribose ring oxygen forms an H-bond to the main chain of Gly 93 . The 3Ј-hydroxyl H-bonds with the main chain nitrogen of Gly 28 , as well as with the side chain of Ser 27 . The NADPH pyrophosphate group has the expected close approach to the tight turn (␤-1 to ␣-2) containing the signature nucleotide binding motif GXXGXXG.…”

Human biliverdin reductase: A member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity

“…The pGEX 4T-2͞human BVR vector was prepared as described in ref. 1. Mutant variants of both pGEX 4T-2 and pcDNA3 constructs were obtained by using a QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using PAGE purified primers, and extensively characterized by restriction analysis, PCR, and DNA sequencing.…”

Biliverdin inhibits activation of NF‐κB: Reversal of inhibition by human biliverdin reductase

“…23 BVR is known as a biliverdin-converting enzyme; however, recent findings have uncovered additional functions of the human reductase that relate to its kinase activity and reflect its structural features. 24 The S/T/Y (serine/threonine/tyrosine) kinase activity of hBVR has been linked to its function in the insulin/IGF receptor signaling cascade in both the MAPK and PI3K branches, 25,26 and its DNA binding has been shown to involve its bZip domain. [27][28][29] hBVR, as with other members of this family of AP-1/CRE binding transcription factors: c-Jun, c-Fos, c-Myc and CREB/ATF-2, is activated by free radicals.…”